A surprisingly small amount of information is known about the specific mechanism of action of GCM2, the essential parathyroid transcription factor; information that is critical for identifying new, potential drug targets for parathyroid gland diseases. The function of the parathyroid glands-to produce parathyroid hormone (PTH)- is essential for maintaining normal calcium homeostasis and bone health. Diseases of the parathyroid glands represent some of the most common endocrine disorders, including hyperparathyroidism, which is primary or secondary to chronic kidney disease. Mutations in GCM2 that cause hypoparathyroidism, and the results of GCM2-/- mice, reveal that GCM2 is a master regulator of parathyroid gland development. It is expressed not only during embryonic development, but also in the mature organ. How does this unique transcription factor direct and maintain the parathyroid gland-specific program? Traditional methods have not been able to address this question. The largest hurdles to understanding this mechanism have been the lack of an available parathyroid cell line and the absence of potent GCM2 antibodies needed to study protein-protein interactions. To circumvent these obstacles, we have established a novel knock-in mouse model carrying a double-tagged GCM2, which preserves the normal behavior of GCM2. This unique in vivo tool will now make possible the identification of target genes and cell-specific binding partners. Our long-term goal is to target components of the GCM2 transcriptional network in order to treat diseases of the parathyroid glands. Supported by our preliminary data, our central hypothesis is that GCM2 works through regulating promoters and enhancers of a network of cell-specific target genes and by binding to specific protein partners. The objectives of this proposal are to systematically identify the target genes of GCM2 and its binding partners and to further characterize the mechanism of action of these components. To approach these goals, we specifically propose to (1) determine DNA binding sites and target genes for GCM2 in the parathyroid gland using parathyroid tissue from our knock-in mouse model. We will be using ChIP-seq and DNase-seq techniques followed by the initial functional analysis of identified target genes. Furthermore, we will (2) identify binding partners of GCM2 using co-immunoprecipitation and tandem mass spectrometry, and perform their initial functional characterization. The contribution of this research will be significant because the action of this key parathyroid regulator is currently a black box - target genes and other players are mostly unknown. The approach suggested in this application, namely the use of an epitope-tagged knock-in mouse model, is innovative, because it focuses on a novel approach, using an epitope-tagged knock-in model instead of the slow and inefficient existing candidate approach. Based on the central role of GCM2 for the parathyroid glands, our results are expected to have an important positive impact; in addition to advancing the field of parathyroid gland biology, the identified components are highly likely to provide new targets for therapeutic interventions.